Myocardial infarction (MI) is responsible for 220,000 deaths annually in the USA [35]. Cardiac ischemia- reperfusion (IR) injury is the pathologic process that links MI to contractile dysfunction. Mitochondrial Ca2+ overload and excessive generation of reactive oxygen species (ROS) are key pathologic events in cardiac IR, leading to opening of the mitochondrial permeability transition (PT) pore and subsequent death of cardiomyocytes. In contrast mitochondria are also implicated in the mechanism of ischemic preconditioning (IPC), in which short non-lethal periods of ischemia protect the heart from prolonged IR injury. An additional factor implicated in IPC is nitric oxide (NO), and we have recently identified two novel mechanisms of NO signaling at the mitochondrial level which are of potential importance in IPC: (i) reversible inhibition of the respiratory chain by NO dependent protein S-nitrosation; (ii) mild uncoupling (H+ leak) mediated via nitrated lipid activation of uncoupling proteins (UCPs) or other targets. Both these events can subsequently inhibit mitochondrial Ca2+ overload and ROS generation, and thus inhibit PT pore opening. To exploit this protective pathway a series of mitochondrially-targeted NO donors have been developed, which exhibit potent cardioprotective effects in cardiomyocyte and perfused heart models of IR injury. Based on these findings, it is hypothesized that reversible respiratory chain inhibition and small regulated increases in H+ leak are mechanisms of NO mediated cardioprotection. This hypothesis will be tested through pursuit of the following specific aims: Aim 1: Test the hypothesis that respiratory chain S-nitrosation mediates the cardioprotective effects of NO. Aim 2: Test the hypothesis that H+ leak activation by nitro-lipids mediates the cardioprotective effects of NO. Aim 3: Test mitochondrially-targeted NO donors as cardioprotective agents. It is anticipated that addressing Aims 1 & 2 will define new mechanisms by which NO mediates cardioprotection, and that Aim 3 will lead to novel therapies for the treatment of cardiac IR injury.